The detection of biologically important thiols such as cysteine and homocysteine has been the focus of much research. Most reported methods have been based upon nonspecific redox chemistry, immunoassays, or upon derivatization with chromophores or fluorophores.
There is an unfilled need for a simple, rapid, accurate, and inexpensive method to assay homocysteine, including, but not limited to, homocysteine in plasma. There is an unfilled need for a simple, rapid, accurate, and inexpensive method to assay cysteine, including, but not limited to, cysteine in plasma. Ideally, such methods would employ stable, non-toxic reagents that afford high sensitivity and selectivity.
At elevated levels in plasma, homocysteine (Hcy) is a risk factor for Alzheimer's and cardiovascular diseases. Current methods for the direct detection of Hcy suffer from interferences caused by other common thiols, such as cysteine (Cys) and glutathione (GSH). Hcy analyses have thus typically been based on chromatographic separations or immunoassays. While such methods can be effective, it would be beneficial to have a simple, rapid method for assaying homocysteine, one that does not require the use of chromatographic or immunological techniques.
Cysteine deficiency has been implicated in conditions including slowed growth, hair depigmentation, edema, lethargy, liver damage, muscle and fat loss, skin lesions, and weakness. Again, interferences from other thiols are a concern. While there are a number of dyes that will react with thiols generally, with a resulting change in color, to the inventors' knowledge there have been no prior reports that any dye would react selectively with Cys or Hcy, or with both Cys and Hcy selectively over other thiols. It is believed that the present invention represents the first example of selective reaction with Cys or Hcy, or with both Cys and Hcy selectively over other thiols. The determination of specific thiols has often been carried out in conjunction with HPLC or capillary electrophoresis separations or via immunoassays.
Hcy has been reported to inhibit the oxidation of luminol and dihydrorhodamine by strong oxidants. Hcy also rapidly reduces ferrylmyoglobin to metmyoglobin. In one study of the ability of GSH, Cys, and Hcy to reduce dehydroascorbic acid (DHA), Hcy caused substantially greater reduction of DHA than did either Cys or GSH. Furthermore, Hcy can cause such reduction at Hcy concentrations more than an order of magnitude lower than those required for GSH and Cys. Hcy thus functions as a potent reducing agent in biological systems, although it may also be responsible for oxidative stress.
Biological thiols are characterized by a delicate balance between their oxidizing and reducing functions. Oxidizing thiyl radicals will rapidly equilibrate to reducing, captodative α-amino carbon-centered radicals under physiological, aerobic conditions. Additionally, reducing disulfide radical anions rapidly decay to the reducing α-aminoalkyl radicals. The equilibria in the free radical chemistry of biological thiols are pH-dependent, and include several radical and recombinant species.
Different naturally-occurring thiols, which may have similar structures, may have quite different physiological properties. The physiological effects and correlations that have been observed for these thiols are a public health concern. An improved ability to detect and quantitate low molecular weight biological thiols would be of great importance to diagnosing and understanding disease states.
Examples of low molecular weight thiols that have more-or-less similar structures, but that have disparate physiological properties, include cysteine, homocysteine, glutathione, N-acetylcysteine, and penicillamine. Generic methods for detecting thiols do not readily distinguish among such similar species. There is a substantial need for improved methods for detecting and quantitating biological thiols.
Thiols are easily oxidized. Many have similar structures. They are typically colorless and non-fluorescent at visible wavelengths. Current detection methods are often tedious. One current method is based on making derivatives with chromophores or fluorophores; but the products can be unstable, and the derivatization reactions often produce interfering byproducts. The relatively large excess of glutathione that is typically present in millimolar concentrations in biological media complicates the detection of other thiols.
The universal methylating agent S-adenosylmethionine (SAM) is synthesized from methionine and ATP. SAM is essential for one-carbon metabolism. Methylation via SAM produces S-adenosyl homocysteine (SAH). This reaction is followed by the enzymatic hydrolysis of SAH by S-adenosyl homocysteine hydroxylase (SAHH) to yield adenosine and Hcy. A trans-sulfuration pathway leading from Hcy to Cys commences at this point. Hcy reacts with serine via cystathionine-β-synthase (CBS), a vitamin B6-dependent enzyme, to produce cystathionine. Cystathionine then reacts to form cysteine, a source of glutathione, sulfate, and sulfite.
But the synthesis of cystathionine is not the only potential fate of Hcy. Homocysteine can also be methylated, released into the extracellular medium, or deaminated. Hcy methylation to methionine can be carried out by methionine synthase in a folate-dependent manner, or via betaine homocysteine methylase.
When Hcy metabolism is disrupted, the export of Hcy from within cells to the extracelluar medium becomes imbalanced, and hyperhomocysteinemia can result. At lower Hcy levels, export rates to plasma and urine are elevated. Higher Hcy levels in plasma and urine are directly related to lower methionine synthase activity, and to folate or vitamin B12 deficiency. “Hyperhomocysteinemia” is often defined as a condition in which plasma Hcy concentration exceeds 14 μM. It has been proposed that vitamin or folate therapy may be useful in treating hyperhomocysteinemia-related disorders.
In blood or plasma, Hcy may bind to other molecules. Approximately 99% of Hcy in plasma binds via disulfide linkages to proteins, other Hcy molecules, or other thiols. Oxidation to a disulfide in plasma is coupled to O2 reduction, leading to oxidative stress. Reactive oxygen species (ROS) levels can be diminished by peroxidases. However, hyperhomocysteinemia appears to inhibit the expression of peroxidases.
Nitric oxide (NO) released by endothelial cells can react with Hcy to produce S-nitrosohomocysteine (SNOHO), a strong antiplatelet and vasodilator agent. A consequence of nitrosylation is to repress peroxide production, and thereby to inhibit ROS formation. However, Hcy is not effectively deactivated by this mechanism at Hcy levels typical of hyperhomocysteinemia.
Low-density lipoprotein oxidized by ROS suppresses endothelial nitric oxide synthase expression. Hcy is believed to lower NO availability upon its own nitrosylation. NO is a neurotransmitterthat is involved in muscle relaxation and microphage cytotoxicity. Lowered NO availability may be among the physiological effects of hyperhomocysteinemia. More importantly, Hcy impairs endothelial cell function in the absence of NO. Although the mechanism is not well understood, it is believed that the direct action of homocysteine on endothelial cells could either involve enhanced oxidative stress, or it could result from the direct effect of the oxidation products of homocysteine.
The impairment of endothelial cells by hyperhomocysteinemia is believed to be one cause of cardiovascular disease. It is believed that Hcy can switch the phenotype of endothelial cells from anticoagulant to procoagulant. In fact, homocysteine-mediated cardiovascular risk may be as high as the risk from hyperlipidemia. High homocysteine levels have been detected in up to 20% of people suffering from heart disease.
It is also known that the impairment of endothelial cells can result in the vasomotor dysregulation that causes Raynaud's syndrome. A recent study has shown the presence of elevated plasma homocysteine levels in Raynaud's syndrome patients.
Oxidative stress generated by hyperhomocysteinemia may be associated with brain damage and diseases such as Alzheimer's. Recent studies suggest that glutathione peroxidases are overexpressed in Alzheimer patients, linking the disease to oxidative stress in the brain. Elevated levels of plasma homocysteine have also been detected. Conversely, antioxidant supplements have been reported to delay the onset of Alzheimer's-related complications. An increased incidence of birth defects, and renal failure are among other diseases that have also been linked to hyperhomocysteinemia.
If hyperhomocysteinemia can be promptly and properly diagnosed, then the physiological effects of hyperhomocysteinemia may sometimes be reversed, at least in part. Proper diagnosis may help to prevent neural tube defects in pregnancy, ischemic heart disease, stroke, and possibly colon cancer. It has been reported that the risk of heart disease can be reduced by up to 40%. Folic acid supplementation has been recommended for these and related conditions.
For a recent review, see generally H. Refsum et al., “Facts and recommendations about total homocysteine determinations: An expert opinion,” Clin. Chem., vol. 50, pp. 3-32 (2004).
Cysteine is the final product of the trans-sulfuration pathway through homocysteine metabolism. The low water solubility of the disulfide reduces its excretion. It can therefore accumulate in urine (leading to cystinuria), or in various organs of the body (e.g., kidney stones). Low levels of cysteine have been associated with slowed growth, hair depigmentation, edema, lethargy, liver damage, muscle and fat loss, skin lesions, and general weakness.
To the knowledge of the inventors, there are no known prior direct colorimetric or fluorometric methods for the specific detection of biological thiols such as homocysteine or cysteine. Detection methods that have been used for these thiols have included chromatographic separations, immunoassays, enzymatic assays, electrochemical separation and detection, mass spectrometry, and flow injection techniques. Many of these prior detection methods have substantial inherent limitations.
Electrochemical detection is complicated by interference from oxidizable impurities. Electrochemical detection of thiols by capillary electrophoresis (CE) is hampered by the need for precision electrode alignment and isolation of the detector from the separation voltage. Amperometric post-column detection of cysteine and homocysteine also can suffer from low selectivity and high background current, as cysteine exhibits irreversible oxidation requiring a positive overpotential. The small volumes of the separation capillaries used in CE require that the detector be placed in-line to minimize line-broadening. Good sensitivity often requires dual electrode configurations. The stability of the detection cell components can be another concern. Mercury and mercury amalgam electrodes have been used for thiols, but their use is limited due to concerns that include toxicity and poor stability. Chemically modified electrodes require a complex preparation procedure, can exhibit poor stability, and need controlled working conditions.
Fluorescence polarization immunoassays (FPIA) and enzyme immunoassays (EIA) have shown (inter-laboratory) imprecision. FPIA requires long run times and has a low throughput. Enzymes are relatively unstable and expensive, making enzyme-based assays less attractive in spite of their potential for high specificity. Radioimmunoassays are undesirable due to the use of radioactive materials. STE (Substrate-Trapping-Enzyme) technology requires a batch chromatography step and has low precision.
Mass spectrometry (MS) coupled to high performance liquid chromatography (HPLC) requires complex, expensive equipment. Gas chromatography-mass spectrometry (GC-MS) also employs complex equipment, and requires tedious procedures that are not well-suited for routine diagnostic applications. Gas chromatography-electron capture detection and flame photometry detection require tedious sample preparations or high operating temperatures. Trap and Release Membrane Introduction Mass Spectrometry (T&R MIMS) requires time-consuming derivatizations and sophisticated instrumentation, making it not well-suited for routine analyses.
Derivatization of thiols with chromophores and fluorophores has also been used to determine specific thiols, often in conjunction with HPLC separations. Thiol derivatizing agents often contain electrophilic alkylating groups for reaction with sulfhydryl moieties. These agents include iodoacetamides, maleimides, and monobromobimanes (mBrB). These agents are typically non-selective among different thiols, and instead react with thiols generally, as well as other biomolecules. Interferences are therefore a concern. For instance, iodoacetamides will react with histidine, tyrosine, or methionine. Other reagents such as 1,1′-thiocarbonyl diimidazole will derivatize cysteine or penicillamine. Derivatization conditions are often time-consuming and complex, and they can sometimes lead to other problems. Excess derivatization agents must often be removed from the reaction mixture. In some cases, the derivatives are prone to unwanted further reactions. For instance, the products of isothiocyanates and succinimidyl esters with biological thiols have limited stability and undergo further reactions with amines to produce thioureas. Amines have been reported to crosslink the derivatized products of maleimide-based agents. Some thiol-chromophore/fluorophore derivatives are sensitive to light or to hydrolysis. The OPA-Hcy adduct is stable only in dark. On the other hand, mBrB produces fluorescent hydrolysis products. When thiols are derivatized with certain maleimides, hydrolysis peaks are seen at both the beginning and the end of chromatographic elution. Hexaiodoplatinate, on the other hand, produces no hydrolysis products. However, hexaiodoplatinate exhibits a broad reactivity; thioethers, thiazolidines and ascorbic acids are among the reported interferences.
Some derivatization agents themselves are prone to instability. Iodoacetamides are unstable to light. In addition, mBrB is photosensitive, and is unstable in water. The instability of certain maleimides in aqueous conditions necessitates the use of water-miscible organic co-solvents.
Commercially available thiol and sulfide quantitation kits use an enzymatic reaction to release thiols, followed by their determination with Ellman's reagent. However, enzymes are expensive and fragile.
Methylviologen (MV2+) is the ammonium dication:

MV2+ has been used as an oxidant in an investigation of the equilibrium kinetics of both the reducing disulfide and the α-amino carbon-centered radicals derived from Hcy, Cys and GSH. Reducing radical formation was monitored via changes in the UV-Vis spectra of solutions containing the methylviologen radical cation that formed in the presence of the biological thiols. See R. Zhao et al., “Kinetics of one-electron oxidation of thiols and hydrogen abstraction by thiyl radicals from α-amino C—H bonds,” J. Am. Chem. Soc., vol. 116, pp. 12010-12015 (1994); and R. Zhao et al., “Significance of the intramolecular transformation of glutathione thiyl radicals to α-aminoalkyl radicals. Thermochemical and biological implications,” J. Chem. Soc., Perkins Trans., vol. 2, pp. 569-574 (1997) It was surmised that formation of the reducing α-aminoalkyl radical derived from Hcy should be particularly favorable, due to an intramolecular hydrogen abstraction mechanism involving a five-atom ring transition state (See FIG. 1(a)). By contrast, in the case of either Cys or GSH, H-atom abstraction to a reducing carbon-centered radical would proceed via less-favored four-membered ring (FIG. 1(b)) or nine-membered ring (not shown) transition state geometries, respectively. See FIGS. 1(a) and 1(b), depicting the inferred proton abstraction leading to formation of the α-aminoalkyl radical from the thiyl radicals of Hcy and Cys, respectively. These references did not describe any appreciable colorimetric selectively among homocysteine, cysteine, and glutathione.
Zhao et al. (1994) and Zhao et al. (1997) both describe procedures conducted in the absence of atmospheric oxygen. For example, Zhao et al. (1997) at page 570 states: “The solutions were deoxygenated by bubbling with Ar gas, and subsequently saturated with N2O, Since oxygen was suspected to be critical, the purging gas was bubbled through an alkaline pyrogallol solution to reduce the oxygen level as much as possible.”
T. Inoue et al., “Determination of thiols by capillary electrophoresis with amperometric detection at a coenzyme pyrroloquinone modified electrode,” Anal. Chem., vol. 74, pp. 1349-1354 (2002) describes the use of a chemically-modified electrode to detect and determine thiol-containing compounds following capillary electrophoresis separation. The solutions used in the analysis were deoxygenated with either an argon purge or a nitrogen purge.
P. White et al., “Electrochemically initiated 1,4 additions: A versatile route to the determination of thiols,” Analytica Chimica Acta, vol. 447, pp. 1-10 (2001) discloses the electrochemical generation of quinoid intermediates and their subsequent reaction with sulfhydryl thiols as a method for quantifying thiols. The solutions used were degassed and stored under argon. All solutions were generally used within one hour of preparation to minimize losses from aerial oxidation.
T. O'Shea et al., “Selective detection of free thiols by capillary electrophoresis—Electrochemistry using a gold/mercury amalgam microelectrode,” Anal. Chem., vol. 65, pp. 247-250 (1993) discloses a method for the detection of thiols by the catalytic oxidation of mercury in the presence of thiols. Deoxygenation was said to be important for reproducibility of the response. The buffer reservoirs were deoxygenated by sparging with argon.
Colorimetric and fluorometric methods for the analysis of carbohydrates are disclosed in U.S. Pat. No. 6,534,316.
A review of current methods for determining homocysteine is given in O. Nekrassova et al., “Analytical Determination of Homocysteine: A Review,” Talanta, vol. 60, pp. 1085-1095 (2003). The drawbacks of current techniques are highlighted in this review. At page 1093 the authors acknowledged that “to date the reaction protocol has not been developed for the selective determination of homocysteine . . . . ” At page 1094 the authors concluded that the existing “techniques have been shown to produce various advantages in terms of sensitivity depending on the conditions required, however, the lack of selectivity inherent in so many of the procedures means that there is always the need for a separation technique to be utilised before detection can occur. This often can add expense and delay in the sample analysis, but has the advantage of producing both a selective and sensitive detection process. Therefore, the ultimate aim would be to produce a device capable of producing both a sensitive and selective analysis with minimal sample pre-treatment and ability to quantitate 1 μM differences.”
There remains an unfilled need for improved, simple methods for determining homocysteine and cysteine.
The present invention has successfully achieved what the authors of the Nekrassova et al. article characterized as “the ultimate aim” in this field. The present invention is based on a simple calorimetric assay. Qualitatively, the assay may be conducted without any instruments at all, relying just upon a visual inspection of color. If quantitative results are desired, they may be obtained, for example, with instrumentation that need be no more complexthan an ordinary absorbance or fluorescence spectrometer.